This disclosure relates generally to methods for sample preparation and analysis utilized within high-throughput screening assays. More specifically, the method is directed at improvements in regulating sample composition for screening assays for use within a nanocalorimeter.
In recent years, researchers and companies have turned to combinatorial methods and techniques for synthesizing, discovering and developing new compounds, materials, and chemistries. For example, pharmaceutical researchers have turned to combinatorial libraries as sources of new lead compounds for drug discovery. Consequently, there is a need for tools that can measure reactions and interactions of large numbers of small samples at high rates, consistent with the needs of combinatorial discovery techniques. Preferably, users desire that these tools enable quick, inexpensive measurements and minimize contamination and cross-contamination problems. In addition there has been an explosion in the number of potential drug targets due to the accelerated implementation of genomics technologies and the completion of the Human Genome sequence.
To further illustrate the use of combinatorial chemistry methods and the need for improved methods, we now discuss the example of pharmaceutical research in this area in more detail. Pharmaceutical researchers have turned to combinatorial libraries as sources of new lead compounds for drug discovery. A combinatorial library is a collection of chemical compounds that have been generated, by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” as reagents. For example, a combinatorial polypeptide library is formed by combining a set of amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can theoretically be synthesized through such combinatorial mixing of chemical building blocks.
Once a library has been constructed, it must be screened to identify compounds, which possess some kind of biological or pharmacological activity. For example, screening can be done with a specific biological molecule, often referred to as a target molecule that participates in a known biological pathway or is involved in some regulatory function. The library compounds that are found to react with the targets are candidates for affecting the biological activity of the target, and hence a candidate for a therapeutic agent.
Since combinatorial methods involve looking at a large number of compounds and reactions, there is a need for tools that can measure reactions and interactions of large numbers of small samples at an accelerated measurement rate, consistent with the needs of combinatorial discovery techniques. Preferably, users desire that these tools enable inexpensive measurements and minimize contamination and cross-contamination problems.
One method for measuring reactions and interactions is calorimetry. Calorimetry can be used to measure the thermodynamics and kinetics of reactions without requiring that reactants be labeled (e.g., radio-labeled or labeled with fluorophores) or immobilized on surfaces. Most other current methods require some modification of either the substrate or a cofactor (fluorescent labeling, surface anchoring, etc.) [Handbook of Drug Screening, R. Seethala and P. B. Fernandes, eds., Marcel Dekker Inc., 2001]. These modifications add steps and cost to an assay, and unless considerable effort is expended to develop a non-interfering assay, they can potentially modify the reagents in undesired ways that may not be understood at the time of an assay.
In some cases, the sample to be studied is precious, and it might not be acceptable to use the relatively large amount of material required by a standard microcalorimeter to perform only one measurement. For example, one may desire to study a natural extract or synthesized compound for biological interactions, but in some cases the available amount of material at concentrations large enough for calorimetry might be no more than a few milliliters. Performing a measurement in standard microcalorimeters, such as those sold, for example, by MicroCal® Inc. (model VP-ITC) or Calorimetry Sciences Corporation® (model CSC-4500), requires about 1 ml of sample, which means that one would possibly be faced with using a majority or all of the precious material for one or a small series of measurements. Tools that enable calorimetric measurements with much smaller sample sizes would be helpful in overcoming this limitation.
A variety of measurement approaches has been used to screen combinatorial libraries for lead compounds, one of which is the competitive binding assay. In this assay, a marker ligand, often the natural ligand in a biological pathway, is identified that will bind well with the target molecule. The assay often requires the chemical attachment of a fluorescent molecule to this marker ligand, and it is important that the fluorescent molecule does not affect the manner in which the marker ligand reacts with the target molecule. Alternatively, the ligand could be radioactively labeled or labeled with a chemiluminescent molecule.
To provide an illustrative example, one approach to operating a competitive binding assay utilizes a target molecule, which is exposed to a mixture of test ligands and a marker ligand, often in microtitre wells. After a time for reaction, the wells are rinsed such that free marker ligand is washed away. In wells where the target molecule and the test ligand are strongly bound relative to binding of the marker ligand, the test ligand has blocked the active site of the target molecule so the marker ligand is not bound and is washed away. Conversely, in wells where the target molecule and test ligand do not bind strongly relative to binding of the marker ligand, the marker ligand binds to the target molecule, at least to some extent, and is therefore not washed away. By investigating the wells for the presence of fluorescence after the washing, reactions of test ligands and target molecules can be determined as having occurred in wells where reduced fluorescence is observable relative to control wells to which no test ligands have been added.
However, competitive binding assays require time and expense to develop the labeled reagents and assay. The principal components that need development are discovering a marker ligand and attaching a fluorophore to the marker in a manner that does not affect its reaction with the target molecule. Attaching the fluorescent marker can often take 3 months of development or more and cost $250K or more once the marker ligand is identified.
An alternative approach is described in U.S. application Ser. No. 10/114,611, filed Apr. 1, 2002, titled “Apparatus and Method for a Nanocalorimeter for Detecting Chemical Reactions”. In this approach, two drops, each containing different reactants, are merged together, and the resulting heat evolution is detected. The signal is detected relative to a reference signal, resulting in detection of the net heat of reaction. However, this approach requires that the two drops have a similar composition, or that the dissimilarities in composition are matched in the reacting drops and the reference system, to keep the heat of mixing from obscuring the heat of reaction. The common mode rejection realized from comparison of the reference and measurement reactions will substantially reduce the contribution of heats of mixing when reference and measurement reactions are well matched. However, problems arise in preparing the necessary solutions to have similar compositions for the purposes of minimizing heats of mixing effects. Since the reactants may have different solvents and co-solvent concentrations when being synthesized and stored, an expensive mixing step and complicated drop-dispensing step are required. An assay method that eliminates the intermediate mixing step required for control of solution composition and simplifies drop dispensing would eliminate this cost and time delay in the discovery process.
Yet another problem is the complexity associated with depositing many different compounds from a compound library at the time of measurement. Since the number of compounds can be large, the control of the delivery of compounds and the need to clean any tips or needles when switching from one compound to another complicates the delivery of reagents to an array such as a nanocalorimeter array. A method that enables pre-formatting of arrays prior to their use and reduces the number of different solutions that need to be delivered or deposited at the time of the measurement would help mitigate this problem.